Summary of Work: Initiation of human DNA replication entails a highly regulated assembly of polymerase complexes along with chaperone proteins and other enzymes. The polymerase alpha with its associated primase activity is the only polymerase involved in this initiation process. This enzyme complex is composed of four proteins, a 180 kDa phospho-glyco protein containing the DNA polymerase activity, a 70 kDa phosphoprotein with no known function, and two smaller subunits of 49 and 58 kDa containing the primase activity. Current research is focused on the enzymatic mechanism and regulation of the two primase subunits. Previously we had cloned and overexpressed the cDNA for the two human DNA primase subunits (Hp49 and Hp58) and purified the proteins from E. coli. Using the overproduced recombinant primase subunit the regulation of the p49/p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase a (pol a) can readily elongate, further primase activity is negatively regulated. This occurs both within the context of the 4 subunit pol a-primase complex as well as the p49/p58 primase complex, indicating that the newly generated primer:template need not interact with pol a in order to regulate further primase activity. Photocrosslinking of single-stranded DNA- primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49/p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer: template binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer:templates suggests that p58 mediates the transfer of primers from the primase active site to pol a. - Biochemistry, Catalysis, DNA Polymerase II, E. Coli, Mice, Nucleotide Mapping, Zidovudine